J Muscle Res Cell Motil (2011) 31:323–336

DOI 10.1007/s10974-011-9238-9

ORIGINAL PAPER

Biomechanics of the sarcolemma and costameres in single skeletal

muscle fibers from normal and dystrophin-null mice
K. P. Garcı́a-Pelagio • R. J. Bloch • A. Ortega •

H. González-Serratos

Received: 26 November 2010 / Accepted: 11 January 2011 / Published online: 11 February 2011
Ó Springer Science+Business Media B.V. 2011

Abstract We studied the biomechanical properties of the Keywords Muscle mechanics  Costamere  mdx 
sarcolemma and its links through costameres to the con- Superficial tension  Dystrophic muscle  Muscular
tractile apparatus in single mammalian myofibers of dystrophy
Extensor digitorum longus muscles isolated from wild
(WT) and dystrophin-null (mdx) mice. Suction pressures
(P) applied through a pipette to the sarcolemma generated a Introduction
bleb, the height of which increased with increasing P.
Larger increases in P broke the connections between the Costameres are structures at the sarcolemma of striated
sarcolemma and myofibrils and eventually caused the sar- muscle fibers that align circumferentially with the Z disks
colemma to burst. We used the values of P at which these and the M bands of the nearest myofibrils. Costameres
changes occurred to estimate the tensions and stiffness of maintain and coordinate the organization of the sarco-
the system and its individual elements. Tensions of the lemma with the underlying contractile apparatus, while
whole system and the sarcolemma, as well as the maximal ensuring that distortions in the sarcolemma during isotonic
tension sustained by the costameres, were all significantly contractions, that lead to shortening below the equilibrium
lower (1.8–3.3 fold) in muscles of mdx mice compared to length, are small and periodic (Bloch et al. 2002; Bloch and
WT. Values of P at which separation and bursting occur- Gonzalez-Serratos 2003; Anastasi et al. 2008). They also
red, as well as the stiffness of the whole system and of the organize membrane domains at the sarcolemma that are
isolated sarcolemma, were *2-fold lower in mdx than in enriched in proteins, signaling molecules, ion channels and
WT. Our results indicate that the absence of dystrophin pumps, that are essential for proper physiological function
reduces muscle stiffness, increases sarcolemmal deforma- of striated muscles (Brenman et al. 1995; Williams and
bility, and compromises the mechanical stability of costa- Bloch 1999a; Oak et al. 2003; Ervasti 2003). They con-
meres and their connections to nearby myofibrils. stitute an essential structure in the pathway for lateral force
transmission from myofibrils through the sarcolemma to
the extracellular matrix, and ultimately to the tendons
K. P. Garcı́a-Pelagio  A. Ortega
(Bloch and Gonzalez-Serratos 2003). Defects in proteins at
Departamento de Bioquı́mica, Facultad de Medicina,
Universidad Nacional Autónoma de México, Mexico, costameres can compromise muscle force production dur-
DF 04510, Mexico ing contraction either directly, by reducing the efficiency of
lateral force transmission, or indirectly, by increasing the
R. J. Bloch  H. González-Serratos
chances that the sarcolemma will be weakened and dam-
Department of Physiology, University of Maryland, School
of Medicine, 655 West Baltimore St., Baltimore, aged, resulting in degeneration or death of the myofiber
MD 21201, USA (Reed and Bloch 2005; Blaauw et al. 2008). Many mus-
cular dystrophies, associated with a profound weakness and
H. González-Serratos (&)
fragility of myofibers (Barton 2006), have been linked to
Departamento de Fisiologı́a, Facultad de Medicina, Universidad
Nacional Autónoma de México, Mexico, DF 04510, Mexico alterations in costameric proteins such as dystrophin
e-mail: [email protected] (Hoffman et al. 1987; Williams and Bloch 1999b;

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Rybakova et al. 2000; Ervasti 2007), sarcoglycans (Nigro membranes (Rand 1964; Evans and Yeung 1989; Hoch-
et al. 1996; Williams and Bloch 1999a; Lapidos et al. muth 2000; Zhang et al. 2007) including the sarcolemma of
2004), and dystroglycans (Ozawa 1998; Campbell and frog muscle fibers (Rapoport 1972). We examine the bio-
Stull 2003; Ayalon et al. 2008). mechanical properties of murine myofibers with the aim of
Dystrophin is a 427 kDa cytoskeletal protein enriched at determining the tension, stiffness and breaking point of the
costameres that is thought to connect the cytoskeleton to attachments between the sarcolemma and nearby myofi-
integral proteins of the plasma membrane (Porter et al. brils, which are likely to occur at costameres, and how
1992; Ervasti and Campbell 1991, 1993; Williams and these properties are affected by the absence of dystrophin.
Bloch 1999a; Rybakova et al. 2000; Ursitti et al. 2004; We report that the links between the contractile apparatus
Bhosle et al. 2006; Ervasti 2007; Stone et al. 2007), and and costameres at the sarcolemma are stronger and stiffer
through the membrane, to the basal lamina (Ohlendieck in wild type than in mdx myofibers, and further, that the
et al. 1991; Dmytrenko et al. 1993; Winder 1997; Campbell ability of the isolated sarcolemma to resist bursting is
and Stull 2003; Beedle et al. 2007). Dystrophin and the weakened in mdx mice. Preliminary accounts of these
proteins with which it associates at the plasma membrane, results have been published elsewhere (Garcia-Pelagio
termed the dystrophin–glycoprotein complex, have been et al. 2006, 2008).
linked to different forms of muscular dystrophy. In par-
ticular, Duchenne and Becker muscular dystrophies are
caused by mutations in the gene that encodes dystrophin, Materials and methods
leading to its absence (Duchenne) or presence at the sar-
colemma in reduced amounts or with impaired activity Animals
(Becker). Despite extensive study, the function of dystro-
phin and its biomechanical role at the sarcolemma, as well WT mice (C57Bl/10ScSn, n = 30) and mdx (C57-Bl/
as how defects in the protein lead to muscular dystrophy, 10ScSn-DMD-mdx, n = 18) mice, from 7 to 11 weeks of
are still poorly understood (Nowak and Davies 2004; age, were used. WT mice were purchased from The Jackson
Claffin and Brooks 2008). Studies of the dystrophin-null Laboratory (Bar Harbor, ME); mdx mice were raised in the
mouse (mdx) model have shown that skeletal myofibers Central Animal Facility of the University of Maryland,
have disorganized costameres, which resemble those seen Baltimore. Before removal of muscles and dissection of
in biopsies of human muscular dystrophies (Porter et al. myofibers, mice were euthanized by cervical dislocation
1992; Ehmer et al. 1997; Minetti et al. 1998; Williams and without anesthesia. All experiments were in accordance
Bloch 1999a; Reed and Bloch 2005). The location and with institutional guidelines for the care and welfare of
structure of dystrophin suggest that it may play a role in the laboratory animals.
stability, stiffness and organization of the sarcolemma.
Consistent with this, dystrophin mechanically reinforces Isolation of muscle fibers
the sarcolemma, protecting it from the membrane stresses
developed during muscle contraction (Zubrzycka-Gaarn Both EDL muscles were rapidly and carefully dissected
et al. 1988; Petrof et al. 1993; Blaauw et al. 2010).) Fur- under low magnification (93), and placed in Kreb’s solu-
thermore, some authors (Zubrzycka-Gaarn et al. 1988; tion, containing in mM: 135 NaCl, 5 KCl, 1 MgCl2, 15
Rybakova et al. 2000; Hutter et al. 1991) have shown that NaHCO3, 11 glucose, 1 Na2HPO4 and 2.5 CaCl2, equili-
dystrophin is tightly attached to the sarcolemma and that it brated with 95% O2 and 5% CO2 to a pH of 7.0. Small
remains attached to the sarcolemma when the latter is bundles of approximately 100 fibers from the third toe
separated from the myofibrils. The absence of dystrophin in muscle of the EDL muscle were isolated under a dissecting
myotubes prepared from the mdx mouse has also been microscope. The bathing solution was replaced at least 3
linked to a substantial reduction in cell stiffness (Pasternak times with Kreb’s solution without Ca2? and containing
et al. 1995). 15 mM EGTA. Bundles of fibers were transferred to
Here we address the possibility that the absence of relaxing solution, containing in mM: 185 K(C2H5COO),
dystrophin results in a decrease in the transmission of 2.5 Mg(CH3COO)24H2O, 10 imidazole propionate, 2.5
passive force laterally, from the myofibrils to the sarco- Na2ATP, 5 EGTA, pH 7.1. Single fibers were isolated from
lemma, linked to changes in the properties of costameres. the bundles as previously described (Gonzalez-Serratos
We use aspiration with a large bore micropipette to 1971). Dissecting in relaxing solution reduced the possi-
examine the biomechanical properties of the sarcolemma bility of damaging myofibers. Similar preparations have
and costameres of wild type muscle fibers and mdx. This been used to measure biomechanical properties in myofi-
method has been used to measure the surface tension bers isolated from normal or mutant mice (Wieneke et al.
(Mitchison 1953), and the mechanical properties of cell 2000; Shah et al. 2004). The increase in myoplasmic Ca2?

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concentration ([Ca2?]i) during exposure of myofibers to Elastimetry

high K? concentrations, similar to the one we used here, is
brief and transient, lasting only a few seconds, after which We modified the method of Rapoport (1972). Micropipettes
[Ca2?]i decreases to normal values (Caputo and Bolaños were attached to 2 manometers, one filled with Hg and the
1994), so prolonged increases in [Ca2?]i do not occur. Low other with perfluorinated polyether liquid (Miller-Stephen-
[Ca2?]o has no deleterious effect on the biomechanical son, CT), with densities q = 13.5 and q = 1.71 g/cm3,
properties of isolated myofibers (Wieneke et al. 2000). We respectively (Fig. 1). The manometers were connected at
confirmed the latter by monitoring the health of the one end to the micropipette and at the other to a syringe
myofibers throughout our experiments, as described below. piston, and could be used independently. The manometers
were first calibrated to zero suction pressure (P) by con-
General procedure necting the pipette to a 2 mm diameter capillary placed
between the pipette and either one of the manometers. The
Isolated fibers were transferred to an experimental chamber capillary was filled with distilled water and graphite parti-
containing relaxing solution, fixed to a stage of a com- cles, and the tip of the micropipette was filled with bathing
pound Laborlux microscope (E. Leica Microsystems, solution. The tips of the capillary were placed at the level of
Wetzlar, Germany). The optical part of the microscope was the myofibers. Graphite particles in the capillary were
placed on an XY stage so that every part of the muscle cell observed with a compound microscope. Changes in the
could be visualized without disturbing the preparation or height of the manometers produced an inflow or outflow of
the subsequent placement of the glass micropipette. One the solution inside the capillary, resulting in the movement
tendon was clamped against a cover slip at the bottom of of graphite particles. When the graphite particles did not
the chamber; the other tendon was fixed to a Narishige M-2 move, the applied pressure was zero. The existence of good
micromanipulator (Labtron Scientific Products, Farming- seals (no leakage) between the micropipettes and the myo-
dale, NY), used to stretch the fiber. Photomicrographs were fiber’s sarcolemma was explored by applying suction pres-
taken with a digital camera (Nikon D70, NY) through a sure, P, which produced a bleb (Fig. 2). A constant value for
910 eyepiece and 940, N.A. 0.75 water immersion P at a given manometer setting indicated a good seal. If we
objective. The fiber was stretched to the desired average
sarcomere length (SL), measured first by eye and then with
Image J software (NIH, Bethesda, MD). The former mea-
surements were made with a calibrated micrometer posi-
tioned inside a 925 eyepiece. The diameter of the fiber was
measured after SL was set. Experiments were done at 8°C,
controlled by a GB32J36 thermistor (Fenwal Electronics,
Framingham, MA), connected to a temperature controller
(Yellow Springs Instrument Co., Inc, Ohio OH), which
maintained the temperature through a two Peltier modulus
(Midland Ross, Cambridge, MA). A micropipette (see
below) was placed on the surface of the myofiber, and
negative suction pressure (P) was applied.

Micropipettes
Fig. 1 Experimental arrangement. The experimental system consisted
Micropipettes were made as described by Fonbrune of (A) a set of manometers, (B) the optical system (not shown) with a
compound microscope to observe the isolated fiber, and (C) the bleb
(1949). Capillary tubes of 2 mm internal diameter were formed by the applied negative pressure P to the myofiber. The syringe
pulled to create a flat tip of internal opening of 0.50–0.66 (1) was connected to manometers filled with perfluorinated polyether
times the diameter of the myofibers. In order to acquire manometer (4) or with mercury (5), which were used independently.
precise measurements of the internal diameter of the We used 4 way key valves (2 and 3) to set P inside the syringe with
either of the manometers. Also shown are the micropipette (6) and the
micropipettes they were not fire polished because the surface of the isolated myofiber (7), attached to the bottom of the
curvature tip of these pipettes introduces microscopic experimental chamber with a clip (8). We induced a bleb of variable
distortions. The micropipettes do not damage the sarco- height h in the surface of the fiber by applying suction pressure. When
lemma. The micropipette was connected to a set of key valve 3 was open to the syringe through valve 2 (shown as closed to
manometer 4 in the drawing) the syringe, manometer and micropipette
manometers (see below) and was positioned on the sur- had the same negative P. By closing the branch between key valve 3 and
face of the myofiber with a micromanipulator (Leica the syringe (as shown in the figure), P inside the manometer and the
Microsystems Inc., Bannockburn, IL). micropipette were the same and remained constant

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Fig. 2 Induction of a sarcolemmal bleb by increasing suction muscle cell surface developed tension due to the stretching and
pressure. a, b Two different suction pressures were applied sequen- resistance to deformation by bending. c–j Photomicrographs of
tially to the sarcolemma of a WT myofiber isolated from the EDL different blebs forming with progressively increasing pressures.
muscle of a mouse. The pipette diameter was 26.6 lm and the fiber Separation of Mo and Mi with increasing suction pressure, is shown
diameter was 55.1 lm. The fiber was stretched to an average SL of in muscle fibers from WT with a SL = 3.3 lm (c–f) and mdx mice
3.2 lm. a The sarcolemma (Mo) and the contractile elements in the with SL = 3.4 lm (g–j). P 9 103 in dynes/cm2 is indicated in the left
myoplasm (Mi) remained attached to each other as the bleb was lower corner of each photomicrograph. The results indicate that, at
sucked into the pipette at a pressure, P = 27 9 103 dynes/cm2. b At a similar SLs and Ps, the distensibility of the WT muscle surface was
higher suction pressure, P = 235 9 103 dynes/cm2, Mo was sepa- considerably lower than that of mdx muscle. The transparent lines
rated from Mi. As a result, the contractile apparatus lay well below the outside the surface of the bleb are due to Becker lines and therefore do
full height of the bleb within the pipette. The results show that the not represent real structures

failed to obtain a constant value for P, we moved the checked the stability of P, except when the sarcolemma burst
micropipette to another spot on the surface of the myofiber and P dropped slightly. We also periodically checked for the
or we replaced the micropipette. If neither of these was effectiveness of the seals by increasing and decreasing P.
effective, we discarded the myofiber. We continuously Typically, in myofibers in which the sarcolemma maintains

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its links to the underlying contractile apparatus, the height of A 450

a bleb (h) increases as P increases, and decreases as P
decreases, with no change in the slope of the P–h curve (no 400
hysteresis). Such curves were taken as additional evidence
350
of good seals (Fig. 3).
The elastic model proposed by Rapoport (1972) ana-
300
lyzes the elastic behavior of the distortion and tension

(dyne/cm2)
lines formed by myofibrils and the sarcolemma in

P x 103
250
response to suction pressures, P, applied over a small area
as a bleb is formed. We followed a similar treatment to 200
analyze our experimental results. P in dyne/cm2, applied
150
to the outer surface of the myofiber was calculated from
P = qghman, where q is the density of the manometer
100
fluid in g/cm3, g = 981 cm/s2 and hman is the difference
of levels in the manometer relative to P = 0 (Taylor 50
2005). We assumed that at equilibrium, when the height
of the bleb becomes constant, the pressure inside the cell 0
0 5 10 15 20
is the same as the suspending medium, whether this
occurs before or after separation of the sarcolemma from h(µm)
the underlying structures. For each applied P, the height B 600
of the bleb formed inside the suction pipette, h, was
measured in lm. At the SLs used for our studies, the
500
diameters of the muscle fibers did not change noticeably
as P increased and the bleb enlarged. As the total length
of the myofiber does not change, the total cell volume 400
therefore remains constant.
(dyne/cm2)
P x 103

Once h was measured and P calculated, the average

300
surface tension, c in dyne/cm, was calculated, as follows:
 
1 1
P¼c þ ðdyne=cm2 Þ ð1Þ 200
r1 r2
where r1 and r2 are the radii of curvature of an asymmetric
100
bent shell membrane. However, since the bleb is part of a
symmetric sphere, then the centers of curvatures are the
same. Therefore, r1 = r2 = r, the single radius of curvature 0
0 5 10 15 20 25 30 35
(Rand 1964; Nelkon 1979; Pellicer et al. 2000). Thus
h(µm)
2c
P¼ ðdyne=cm2 Þ ð2Þ
r Fig. 3 Pressure–displacement curve and effect of SL in a WT muscle
fiber. a P was first increased in steps (solid lines) and then decreased
The radius, r, was computed from r = d2/8 h ? h/2 (dashed lines). S denotes the pressure at which separation of the
(lm) from Pythagoras’ Theorem (Dull 1941), where h and sarcolemma from the underlying contractile structures, Psep, occurred
the pipette diameter d were measured. So substituting r in (point 17). Insets Photomicrographs of the bleb: (a) point 4, without
separation of the sarcolemma, Mo, from the contractile elements, Mi.
Eq. 2, it becomes: (b) point 17, at which Mo separated from Mi. (c) point 19, at which
2c pressure was further increased after separation occurred. The results
P¼ d2

ðdyne=cm2 Þ ð3Þ show hysteresis after point 19. b Separation of the sarcolemma from
8h þ h2 the contractile elements depends on SL. Pressure–displacement
curves were generated in different WT myofibers, each of which
From the P–h curves the tension of the whole system, was stretched to the SL indicated in the left upper corner of the figure.
ci?o (dyne/cm), before separation of the sarcolemma, Mo, Filled symbols joined with solid lines are the segments of the curves
from the contractile apparatus, Mi, was calculated from before separation of the sarcolemma, Mo, from the contractile
apparatus in the myoplasm, Mi. Open symbols joined with dashed
Eq. 3. The tension of the isolated sarcolemma after lines are the remaining segments of the curves after separation of Mo
separation from the contractile apparatus, co (dyne/cm), from Mi. The results show that Psep increases as SL increases

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was also estimated with Eq. 3, from the experimental data Therefore, the less elastic the material, the more compliant
in the segment of the P–h curve after separation of the it is. The breaking point is the maximum load that a system
surface membrane from the myoplasm. In this condition, can support before a complete failure of the structure of the
the myofibrils do not contribute to the tension of the material in question. k was estimated from F = kh (dyne)
surface membrane, as measured with the elastimeter. As a (Taylor 2005). Therefore,
result, we were unable to measure the biomechanical F
characteristics of the myofibrils (see Rapoport 1972 for k¼ ðdyne=cmÞ ð6Þ
h
additional considerations).
The attachments between the peripheral myofibrils and Other authors have also calculated cell stiffness (k), by
the sarcolemma are assumed to be due to structures that link measuring the applied force (F) and the displacement it
the myofibrils at costameres. The separation of the sarco- caused on the cell membrane (Petersen et al. 1982; Pasternak
lemma from the most superficial myofibrils takes place when et al. 1995; Pasternak and Elson 1985; Wojcikiewicz and
high pressures (P) are applied to the surface membrane. Zhang 2004).
Since the separation of myofibrils from the sarcolemma As surface tension is inversely proportional to temper-
occurs without breaking either structure, we propose that the ature (Bull 1964), we performed our experiments at a
break takes place in the structures that link the myofibrils to constant temperature of 8.0 ± 0.5°C. The same fiber could
the sarcolemma, i.e. the costameres and closely associated be used for up to 4 h before showing signs of deterioration
cytoplasmic structures. Therefore, the tension exerted by (e.g., spontaneous membrane blebbing, localized swelling,
costameres and closely associated cytoplasmic structures spontaneous localized changes of SL, or lack of repro-
(cc) is obtained by the subtraction of co and ci from ci?o. ducibility of the data obtained from nearby regions of the
same fiber).
ciþo ¼ co þ ci þ cc ðdyne/cmÞ
Thus, cc ¼ ciþo  co  ci ðdyne=cmÞ Data acquisition
Because ci = 0 when the sarcolemma has separated
from the contractile apparatus, it does not contribute to cc. Images were obtained with a digital camera (Nikon D-70
Therefore, NY, USA) with an effective resolution of 6.1 Mpixels. The
camera was placed above the microscope with an external
cc ¼ ciþo  co ðdyne=cmÞ ð4Þ device independent of the microscope and linked instru-
The myofibrils, costameres and associated structures, and mentation, to avoid transmission of vibrations. Several
sarcolemma were considered as three different spring images of the bleb were taken before and after the time at
elements, each with its own elastic properties. The which the height of the bleb was stable, but for the mea-
sarcolemma and myofibrils act as springs in parallel, linked surements described here only the last ones were examined
at right angles by the spring-like elements at costameres. At and used for quantification. Recoverable deformations in
equilibrium muscle length (2 lm/SL), the springs are not membranes have been observed and studied since the
stretched but when the fiber is stretched to [2 lm/SL, the beginning of microscopic observations of cell morphology
tension of each spring increases as a function of the SL. (Rand 1964; Evans and Hochmuth 1976). In our case after
The force, F, in dynes applied to the membrane at a P was applied, it took some time for the bleb to reach a
given suction pressure is estimated from: stable height, suggesting viscoelastic behavior (Rapoport
1972; Garcia-Pelagio et al. 2008), i.e., behavior resembling
F that of an elastic solid on short time-scales and of a viscous
P¼ ðdyne=cm2 Þ ð5Þ
A fluid on long time-scales (Boal 2006). The photographs
Since the radius, r, of the pipette is always the same, we were transferred to a PC with Adobe PhotoshopTM 7.0.1.
can estimate the area, A, of the segment of the spherical The measurements of the height, h, of the bleb formed
shell of the sarcolemma inside the pipette at different Ps. inside the suction pipette were analyzed with Image J
Therefore, F = PA (dyne), where A in cm2 is the area of (NIH, Bethesda, MD). A microphotograph of a stage
the segment of the spherical shell bleb to which the force is micrometer scale, calibrated in microns, was also taken and
applied. A can be calculated from A = 2prh, where r and h transferred to the PC, to determine the relationship between
are defined as above. pixel number and lm.
The stiffness or spring constant (k) is related to the
ability of the cell to resist deformation when subjected to Statistics
load (Leckie and Dal Bello 2009). This also represents the
elasticity of the material under study. Specifically, the more Data for tension, pressure and stiffness were analyzed with
elastic a material is, the less it is deformed by a given force. a 2-way analysis of variance (ANOVA) with a post hoc

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analysis (Kruskal–Wallis). Values of P \ 0.05 were con- pressures, the sarcolemma and the associated contractile
sidered statistically significant. All results are reported as apparatus underlying it were drawn into the pipette toge-
mean ± standard deviation (SD). We also report the sta- ther (Fig. 2a). The movement of contractile apparatus into
tistical population variance (V) of tension and pressure pipette was small compared to the fiber diameter, which
measurements, which indicates the degree of dispersion or minimized changes in volume. Upon relaxation of P, h
scattering of the experimental data. decreased and the bleb returned to its initial height. As the
suction pressure increased, h increased, until at large neg-
Source of materials ative pressures the sarcolemma (Fig. 2b; indicated by Mo)
separated from the contractile apparatus, which remained
Unless indicated, all chemicals were purchased from distended (Fig. 2b; indicated by Mi), even when the pres-
Sigma-Aldrich (St. Louis, MO). sure was relieved. Further increases in P produced further
increases in h but had only minimal effects on nearby
myofibrils (not shown, but see Rapoport 1972).
Results We obtained a series of microphotographs of sarco-
lemmal blebs that were formed with progressively
Elastimetry in mouse myofibers increasing suction pressures in myofibers isolated from the
EDL muscles of WT (Fig. 2c–f) and dystrophin-null, mdx
The elastic properties of the sarcolemma and the strength (Fig. 2g–j) mice. We generated vertical pressure–dis-
of its connections to the underlying contractile apparatus placement (P–h) curves from all fibers, and analyzed them
were assessed by elastimetry. The method uses suction to determine the blebbing behavior of the sarcolemma and
pressure (P), applied over a small sarcolemmal area nearby myofibrils as a function of P. We did not observe
(approximately 250 lm2) through a suction pipette. With significant changes in the SL or nearby myofibrils that
myofibers at an average SL of 2 lm or less, application of contributed to the formation of the bleb and its growth as P
negative pressure to the surface by a large bore suction was gradually increased, suggesting that the sarcolemma
pipette, with a diameter approaching that of the myofiber and nearby myofibrils retained their structural relationship
itself, the sarcolemma and nearby myoplasm were sucked (Fig. 2). The relationship of the myofibrils to the SL did,
into the pipette, without a significant change in surface however, change markedly when P was increased further,
tension. This also occurred at SLs [ 2 lm when we used leading to the separation of these structures (see below).
large bore pipettes. However, at SLs above 2 lm and with
micropipettes with tip diameters of 0.50–0.66 myofiber Vertical bleb displacement, h, is a function
diameter, this did not occur; instead, the sarcolemma of the suction pressure, P
stretched and surface tension (c) increased. These results
are consistent with previous studies of frog myofibers The experimental pressure–displacement (P–h) curve for
(Rapoport 1972), but as the diameters of murine myofibers WT muscle showed three distinct segments (Fig. 3a). In the
are half or less of frog myofibers, our experiments required first, obtained at low suction pressures (Fig. 3a, points
much larger suction pressures, which we achieved with a 2–16), the sarcolemma remained closely associated with
different set of manometers (see ‘‘Materials and methods’’ nearby myofibrils under the bleb. The P–h relationship in
section). At SLs \3 lm the sarcolemma has invaginations this segment was reversible (Fig. 3a, points 8–10 and
and caveolae (Dulhunty and Franzini-Armstrong 1975). 14–16), with no hysteresis when P was reduced. Consistent
During formation of blebs at short SLs, caveolae are with this, morphological observations indicated that the
unfolded as the suction pressure, P, is increased, raising the system formed by the contractile apparatus, costameres,
possibility that, at short SLs, the calculation of the surface and the sarcolemma remained intact.
tension may be misleading. We therefore also routinely In the next portion of the P–h curve (Fig. 3a, points
obtained P–h curves at SLs [ 3.5 lm, where further con- 17–19), measured at higher suction pressures, the slope
tributions of caveolae are likely to be minimal (Dulhunty decreased. This decrease in slope was accompanied by the
and Franzini-Armstrong 1975). We ensured that the values physical separation of the sarcolemma from the myofibrils
we determined were at equilibrium, when the bleb no (Fig. 3a, point S). Thus, in this range the sarcolemma
longer underwent changes in h. distended more for the same increase in pressure, pre-
When we used small bore micropipettes to study sumably because it was not restrained by links to con-
myofibers stretched to SLs [ 3.5 lm, the application of tractile structures.
suction pressure to the surface of the fiber induced the The third segment of the curve (Fig. 3a, points 19–23),
formation of a bleb of variable height, h, which was a obtained by reducing P after separation of the sarcolemma
function of both P and SL (Fig. 2a–j). At low negative from underlying myofibrils, resulted in different values of h

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330 J Muscle Res Cell Motil (2011) 31:323–336

at a given value of P than before separation. Larger with SL, indicating that the contractile apparatus had no
changes in h occurred for the same changes in P, than in the influence on the elasticity of the isolated sarcolemma.
rising portion of the curve. This hysteresis in the P–h curve Psep was also a function of SL, with higher pressures
indicates that the biomechanical system is different before needed to cause separation as SL increased (Figs. 3b, 4a).
and after separation of the sarcolemma. The change in Psep was greatest at SLs from 3.6 to 4.3 lm.
We calculated the surface tension, c, from the slope of the Changes at SLs between 2.9 and 3.4 lm (data not shown)
P–h curve (see ‘‘Elastimetry’’ section, in Materials and were not statistically significant.
methods). For single, isolated myofibers from WT mice, the Consistent with the lower stiffness measured for the
three segments of the curve yielded 3 distinct slopes, for the surface membrane and nearby myofibrils in mdx myofi-
intact system, including the elastic constants (ki?o) of brils, we found that Psep was reduced by 2-fold at SLs
myofibrils, costameres and sarcolemma, for the sarcolemma between 3.6 and 4.3 lm (Fig. 4a; Table 1; P [ 0.05).
immediately after separation, and for the final segment, These results suggest that the sarcolemmal links to the
which reflects the elastic properties of the isolated sarco- myofibrils fibers are significantly weakened in mdx muscle.
lemma (ko). In wild type muscle, the elastic constant of the
sarcolemma alone, ko, was *80% that of the intact system, Elastic behavior
ki?o (Table 1). As the sarcolemma and myofibrils remain
intact after separation, the links between them, established in The sarcolemma and nearby contractile apparatus can be
part at costameres, are the weakest structures in the system. modeled as an elastic body composed of three main ele-
We used the same methods to determine the P–h rela- ments, sarcolemma, costameres and myoplasm, each with
tionship in myofibers isolated from mdx mice. Although the its own elastic characteristics that together generate the
shapes of the curves were similar, the values we obtained elastic properties of the whole system. We first character-
for the pressure at which separation occurred, Psep, for ized the elastic properties, tension and stiffness, of the
surface tension, c, and for ki?o and ko, were consistently whole system and those of the costameres and sarcolemma,
*2-fold lower than in WT muscle (Table 1), suggesting in WT mice and then compared the results with those
that the stiffness of the system encompassing the sarco- obtained with mdx mice.
lemma, costameres and nearby myofibrils, and the strength Using the Young–Laplace equation (Eq. 2) and the data
of the connections between the sarcolemma and myofibrils, in our P–h curves, we computed the total tension of the
are significantly compromised when dystrophin is absent system, ci?o (Fig. 5a), the sarcolemmal tension, co
(Fig. 4a). In the absence of dystrophin, the sarcolemma is (Fig. 5b), and the maximal tension sustained by the
more easily distended at a given pressure, and the slopes of costameres and their links to the myofibrils, cc (Fig. 5c).
each phase of the P–h curves are greater in the WT than in The value for cc was obtained by subtracting co from ci?o
the mdx (Fig. 4b). (Table 1). The total tension sustained by the whole system
and by the isolated sarcolemma in WT myofibers was
The effect of SL significantly larger than for mdx myofibers at all SLs
examined (P \ 0.05). As ci?o increased for WT but not for
The slopes of the rising portions of the P–h curves showed mdx at SLs [ 3.6 lm, these differences become greater.
a strong dependence on SL at pressures at which the sar- Similarly, co for WT was consistently greater than for mdx,
colemma remains attached to the underlying myofibrils. In with the differences reaching significance (P \ 0.05) at
particular, higher pressures were required to produce the SLs between 3.8 and 4.3 lm. The calculated values for cc,
same increase in h (Fig. 3b, solid lines). This suggests that obtained by subtracting co from ci?o, were increased sig-
the contractile apparatus contributes significantly to the nificantly at SLs of C3.4 lm, with differences of *5-fold
stiffness of the sarcolemma in the intact system. This was at longer SLs (Fig. 5c; Table 1).
not the case after sarcolemmal separation, however; these We also examined the variance, V, of our measurements
descending portions of the P–h curve showed little change to assess the variability. The V of measurements of c were

Table 1 Psep, Pbursting, k, ci?o, co and cc, and the population variances, Vci?o, Vo and Vcc expressed as mean ± SD, calculated from all SLs
examined
Psep 9 103 Pbursting ki?o ko ci?o Vci?o co Vo cc Vcc
(dynes/cm2) (dynes/cm2) (dynes/cm2) (dynes/cm2) (dynes/cm2) (dynes/cm2) (dynes/cm2)

WT 367 ± 26 631 ± 47 1894 ± 142 1524 ± 96 188 ± 21 46 ± 5 151 ± 25 30 ± 4 37 ± 6 4±1

mdx 171 ± 19* 321 ± 34* 989 ± 204* 846 ± 102* 99 ± 10* 2±1 89 ± 14* 3±1 11 ± 4* 1±1
* Significant difference from control (P \ 0.05 or lower)

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J Muscle Res Cell Motil (2011) 31:323–336 331

Psep We computed the stiffness (k) of the system and its

A 600
elements as described above (Eq. 6) and the slope of the
Force–displacement (F–h) curve (Fig. 5d, e). These values,
500 too, decreased significantly in mdx myofibers compared to
WT (P \ 0.01; Table 1).
400
(dynes/cm2)

Pbursting of the sarcolemma

P x 103

300

Further increases of suction pressure, P, after separation of

200 the sarcolemma from nearby myofibrils, eventually caused
the membrane bleb to burst (Fig. 6a, b). The pressure at
100 which sarcolemmal bursting occurred, Pbursting, was inde-
pendent of SL (Fig. 6c), as expected, but was consistently
0 higher in WT than in mdx myofibers (Fig. 6a; Table 1;
3.0 3.4 3.6 3.8 4.0 4.3
P \ 0.01). This supports the idea that dystrophin stabilizes
SL (µm)
the sarcolemmal membrane, independent of its possible
B 500 contributions at costameres and their links to nearby con-
WT tractile structures.
450
MDX y = 103.8x - 1459.2
400
Discussion
350

300 We used a modified elastimeter to measure the membrane

(dynes/cm2)

y = 45.8x - 200.6
P x 103

bending elasticity of the sarcolemma of mammalian

250
myofibers. A major advantage of this method is that the
200 geometry and tension of the membrane is well controlled
during the experimental measurements, which allowed us
150
y = 20.6x - 94.2
to quantify the changes in membrane area as a function of
100 suction pressure (Evans and Hochmuth 1976; Needham
and Hochmuth 1992), as well as related changes. In
50
adapting a method originally designed for frog myofibers
y = 24.3x - 276.0
0 (Rapoport 1972), which are significantly larger than
0 5 10 15 20 mammalian myofibers, we had to use micropipettes of
h (µm) smaller diameter, which in turn required manometers that
could operate at much higher pressures. Our methods have
Fig. 4 Effect of SL on separation pressure in WT and mdx myofibers.
a Psep was determined for WT and mdx myofibers. Bars indicate
allowed us to study several biomechanical properties of the
mean ± SD. ** indicates values significantly different from each surface of mature fibers of the murine EDL muscle, but
other, P \ 0.01. For all experiments (n = 8 for WT; n = 5 for mdx). they can easily be modified to study cells with very dif-
In WT myofibers, the dependence of Psep on SL was greater than in ferent surface rigidities. Given the high pressures needed to
mdx. b Shift in the P–h curve in WT and mdx myofibers, stretched to
an average SL of 3.5 lm. Filled symbols and solid lines represent data
obtain detachment and bursting, other quantitative meth-
obtained before separation of the sarcolemma, Mo, from the ods, including optical tweezers and atomic force micros-
contractile elements, Mi. Open symbols and dashed lines represent copy, would likely be ineffective. Our results indicate that
the data obtained after separation of Mo from Mi. S denotes the the elasticity and stability of the sarcolemma and the
suction pressure at which separation occurred. The equations of the
regression lines calculated from the equation, y = mx?b, are given
strength of its links to nearby myofibrils are severely
next to each region of both curves. Psep for mdx myofibers was smaller compromised in the absence of dystrophin.
than for WT, indicating a weaker attachment of the sarcolemma to Our experiments are the first to determine the biome-
underlying contractile elements chanical properties of the sarcolemmal membrane and the
links it establishes via costameres to nearby myofibrils. We
achieved this by studying the properties of the sarcolemma
generally greater for WT than for mdx (Table 1). We sug- while it remained attached to the underlying contractile
gest that dystrophin is an elastic element, linking the sar- apparatus, and after it separated from contractile structures
colemma to the contractile myofibers, which stretches as at higher applied pressures. The properties of the isolated
SL is increased. sarcolemma, obtained after detachment at higher pressures,

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Fig. 5 Surface tensions (c) and

stiffness (k) in WT and mdx
myofibers as a function of SL.
The figure shows histograms of
tensions of the total system
(ci?o, a), the sarcolemma (co, b)
and the maximal tension
sustained by the costameres and
associated structures (cc, c; see
text) at different SLs in WT and
mdx myofibers. The mdx fibers
had consistently lower values
for c and showed no dependence
of c on SL. d, e Stiffness was
computed from Hooke’s Law.
d ki?o, stiffness sustained by the
intact contractile apparatus–
costameres–sarcolemma
complex and e ko, sarcolemmal
stiffness, measured after
separation of the sarcolemma.
Bars indicate mean ± SD. Both
values were lower in mdx than
WT muscle fibers. ** indicates
values significantly different
from each other, P \ 0.01 and *
denotes values significantly
different from each other,
P \ 0.05 (n = 7 for WT and
n = 5 for mdx)

were independent of SL. Comparison of the two conditions Our studies are also the first to assess the role of dys-
suggest that *80% of the stiffness of the muscle cell trophin in determining the biomechanical properties of the
surface is attributable to the sarcolemma and closely sarcolemma linked to nearby myofibrils, and of the sar-
associated structures, including the membrane-associated colemma immediately after detachment from contractile
cytoskeleton and the basement membrane. Nevertheless, elements. Our measurements demonstrate that the absence
the stiffness of the myofiber surface, with the sarcolemma of dystrophin in the EDL muscles of mdx mice decreases
connected to underlying contractile structures, is influenced both the pressure needed to detach the sarcolemma from
by the latter, as increases in SL result in significant superficial myofibrils, Psep, and the pressure needed to burst
increases in stiffness (Table 1; Fig. 5d, e). This is consis- the sarcolemma, Pbursting. Our results suggest that the sar-
tent with the idea that much of the contractile force in colemma of muscle fibers lacking dystrophin is more
skeletal muscle is transduced along lateral pathways of compliant and less stable than control muscles. The role of
force transmission (e.g., Street 1983; Bloch and Gonzalez- dystrophin on isolated segments of the sarcolemma agrees
Serratos 2003). with earlier studies that showed that dystrophin associates

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J Muscle Res Cell Motil (2011) 31:323–336 333

sarcolemma and the contractile apparatus that they anchor,

are significantly weaker than the sarcolemma itself, con-
tributing only *20% of the stiffness seen in the intact
biomechanical system. As dystrophin is normally concen-
trated at costameres (Porter et al. 1992; Straub et al. 1991;
Ervasti 2003) it is not surprising that the strength of these
links is compromised in the mdx mouse. Several proteins
are likely to interact with dystrophin to establish these
links, including c-actin, intermediate filaments composed
of desmin and of keratins, as well as other proteins (Ry-
bakova et al. 2000; O’Neill et al. 2002; Stone et al. 2007).
Similar studies of muscles lacking each of these proteins
should reveal how they contribute to the stability of
costameres and the connectivity of the sarcolemma and
contractile elements.
Analysis of the statistical variance, V, of our values for
Psep offers additional insights into the nature of the costa-
meres and their links to the contractile apparatus. The
variance of Psep with SL is considerably lower for mdx than
WT (100 ± 10 for WT, 9 ± 3 for mdx). This indicates that
if an important elastic element, such as dystrophin, is
missing, the system becomes more compliant. When the
surface membrane is attached to underlying myofibrils via
costameres, stretching the links will produce a larger
change in force than when these links are broken, and this
in turn will produce a higher variance.
Fig. 6 Bursting of the sarcolemmal bleb at large negative pressures. Comparison of our results with those obtained in the
Increasing negative pressures were applied to the surface membrane semitendinosus muscles of Rana pipiens suggests that the
of the myofibers after separation of the sarcolemma from the sarcolemma of the latter is weaker than that of the mouse
underlying contractile structures, until the surface membrane burst EDL muscle. At SLs of 4 lm, Rapoport (1972) measured
(arrows). a, b Myofibers from WT (a) were studied at SL = 3.2 lm
and from mdx mice (b) at SL = 3.3 lm. Values for P 9 103, in dyne/ an average c = 45 dyne/cm for the frog semitendinosus.
cm2, at which each photomicrograph was taken, is given at the Our values are 3–5-fold higher. Similar differences are also
bottom of each image. c Histograms of bursting pressure Pbursting as a found at shorter SLs. Although some of the difference may
function of SL. At all SLs studied, Pbursting of myofibers from WT be due to temperature (22°C, compared to 8°C, used here),
mice were approximately 2-fold higher than in mdx myofibers. Bars
indicate mean ± SD; ** indicate values significantly different from which affects surface tension (Bull 1964), studies of single
each other, P \ 0.01 (n = 5 for WT and n = 3 for mdx) EDL fibers at different temperatures indicate that the Q10
for total surface tension is *3.9 (not shown), which is not
sufficient to account for the different values for c in frog
with the sarcolemma when it is mechanically pealed away and mouse muscles. The higher total surface tension of the
from the contractile apparatus (Rybakova et al. 2000). We EDL myofibers may be due to the presence in mammalian
cannot rule out the possibility that the absence of dystro- striated muscle of costameres that link superficial myofi-
phin leads to other, secondary changes at the sarcolemma brils to the sarcolemma at the levels of the Z-disks and
or the extracellular matrix, including the basal lamina, M-bands, and that may also include longitudinal elements.
collagen fibrils, or other proteins of the reticular lamina By contrast, amphibian semitendinosus myofibers have
that may affect these properties. Nevertheless, our results costameres only at the level of Z-disks (Street 1983;
suggest that dystrophin stabilizes the sarcolemma and Gonzalez-Serratos, unpublished results). The presence of
participates in the links between that membrane and the more links in EDL muscles would produce stiffer surface
contractile apparatus, anchored at costameres. membranes and larger surface tensions. As a substantial
We were able to deduce some of the biomechanical percentage of active force is transmitted laterally through
properties of costameres by comparing the properties of the the costameres to the tendons (Street 1983; Bloch and
sarcolemma in two configurations, attached and detached Gonzalez-Serratos 2003), the higher density of costameres
from the underlying contractile apparatus. Our results in mammalian muscle may explain why a single murine
suggest that costameres, and the links between the flexor digitorum brevis myofiber generates a specific force

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334 J Muscle Res Cell Motil (2011) 31:323–336

of 362 kPa (Allen et al. 2008), compared to 275 kPa for an smaller difference is probably due to the fact that sarcoballs
isolated amphibian muscle cell (Gordon et al. 1966), when are spheres composed of the sarcolemma alone, and that
both are measured at optimal SL. many peripheral membrane proteins, such as dystrophin,
The images of Williams and Bloch (1999a, b) suggest may be shed from the cytoplasmic surface of the sarco-
that costameres cover approximately half of the surface lemma as sarcoballs form.
membrane of fast twitch muscle fibers. At an average SL of Using a different approach, Pasternak et al. (1995)
2.9 or 3.6 lm, the pressure exerted on sarcolemma just reported that the stiffness of myotubes prepared from mdx
before it separates from the underlying contractile appa- mice was 3.4 dyne/cm, several fold lower than the value of
ratus is approximately 2.6 9 109 Pa. If this pressure is 12.3 dyne/cm that they measure for controls. As myotubes
distributed evenly across all domains of the costameres, lack an organized contractile apparatus and do not readily
then the pressure exerted on the links between the con- form costameres (Bloch, unpublished results; but see
tractile apparatus and the sarcolemma, present at costa- Quach and Rando 2006), the fact that they were consid-
meres, would be approximately twice this value, or erably less stiff than myofibers (our values for stiffness are
approximately 5.2 9 109 Pa. We previously estimated that considerably higher) is not surprising. Their use of cells in
during a maximal tetanic contraction the lateral pressure culture prevented Pasternak et al. (1995) from controlling
exerted on the sarcolemma is approximately 6 9 105 Pa SL, and the glass rods they employed to indent the surface
(Bloch and Gonzalez-Serratos 2003). This indicate that the membranes also did not allow them to control for the
costameres have a safety factor of several orders of mag- contribution of caveolae to the membrane as it deformed.
nitude that would make it highly unlikely for them to break Both of these factors also contribute to the lower values
during normal muscle function or even during a maximal they obtained. The higher values that we report for the
lengthening contraction. surface membrane of intact skeletal muscle fibers are likely
Even in their weakened state, in the mdx muscle, due to presence of an extensive membrane-associated
costameres should be more than strong enough to withstand cytoskeleton that underlies the sarcolemma and that links it
damage caused by contractile activity. Thus, damage to the to contractile structures (Porter et al. 1992; Williams and
sarcolemma that occurs during forceful contractions, in Bloch 1999a; Hutter et al. 1991; Na et al. 2009).
mdx as well as in wild type muscle, is likely to arise from Bobet et al. (1998) and Wolff et al. (2006) reported that
areas between the costameres, the ‘‘intercostameric there were no differences in the passive mechanical or active
regions’’, which lack many of the structural proteins that contractile properties of whole EDL muscles from mdx and
reinforce the costameres themselves (Williams and Bloch control mice. It is difficult to compare results obtained with
1999b). The fact that dystrophin and its associated proteins whole muscles to single isolated myofibers, because of the
are present in the intercostameric regions of wild type many additional elastic elements present in the former, some
muscle, but not mdx muscle (Williams and Bloch 1999b), of which increase in mdx muscle (Goldspink et al. 1994). As
may therefore account for the greater susceptibility of the we find that the surface of mdx myofibers is more compliant
latter to damage linked to contractile activity. than controls, the changes in the extracellular matrix (and
Other authors have used aspiration pipettes to measure possibly other structures lying between myofibers) in mdx
cell surface tension (c) in a variety of cells. In neutrophils, mice are likely to compensate for this increased compliance
Needham and Hochmuth (1992) extrapolated the tension in in whole muscles. The fact that the stiffness of the surface of
a resting membrane at room temperature and obtained a mdx myofibers is *100 fold greater than that of WT or mdx
c = 2.4 9 10-2 dyne/cm, similar to the value of 2.7 9 myotubes is consistent with the idea that, despite its
10-2 dyne/cm reported by Tsai et al. (1993). In granulo- importance for the health of muscle and the stability of the
cytes, Evans and Yeung (1989) found c = 3.5 9 sarcolemma, factors others than dystrophin play important
10-2 dyne/cm for granulocytes while Trans-Son-Tay et al. roles in determining the biomechanical properties of the
(1991) reported a value of 2.4 9 10-2 dyne/cm. In chick muscle cell surface. The absence of dystrophin results in a
embryo fibroblasts, Thoumine et al. (1999) and Rand (1964) decrease in the lateral transmission of passive force, linked
found a maximum c = 2.4 dyne/cm, while Waugh and to changes in the properties of costameres. The decrease in
Evans (1979) reported c = 6.6 dyne/cm. Hutter et al. (1991) costameric strength and stiffness in mdx myofibers could
measured the tension of isolated sarcolemmal vesicles alone also lead to a decrease in the lateral transmission of active
(‘‘sarcoballs’’) obtained from semimembranosus muscles by force during contraction (Street 1983; Bloch and Gonzalez-
exposing excised muscles to KCl and collagenase at room Serratos 2003). We are currently studying the roles of sev-
temperature. They reported an average bursting tension of eral other proteins that are enriched at costameres, with the
6 dyne/cm in sarcoballs from myofibers of WT mice and of goal of developing a comprehensive physical model of these
5.2 dyne/cm in sarcoballs of myofibers from mdx mice. The structures and how they contribute to lateral force
difference is smaller than the one we found here. This transmission.

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J Muscle Res Cell Motil (2011) 31:323–336 335

Acknowledgments This research was partially supported by grants Dmytrenko G, Pumplin D, Bloch R (1993) Dystrophin in membrane
to R. J. Bloch from the National Institute of Heath (5R01AR055928) skeletal network: localization and comparison to other proteins.
and the Muscular Dystrophy Association. K. P. Garcı́a-Pelagio was J Neurosci 13(2):547–558
supported during the Ph. D. program (Doctorado en Ciencias Bio- Dulhunty AF, Franzini-Armstrong C (1975) The relative contribution
médicas, Universidad Nacional Autónoma de México) by a scholar- of the folds and caveolae to the surface membrane of frog
ship from Consejo Nacional de Ciencia y Tecnologı́a. skeletal muscle fibers at different sarcomere length. J Physiol
250:513–539
Dull RW (1941) Mathematics for engineers. McGraw-Hill Book
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